... such a popular model organism? Zebrafish (Figure 1) were initially recognized as a useful vertebrate ...deactivation method in zebrafish are short, modified oligonucleotides called morpholinos. When injected into zygotes...CRISPR/Cas9-mediated insertion of foreign DNA. Despite these caveats, several features of zebrafish—including...

...determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV ...protocols, titering methods, and quality control are described below. AAV Production AAV distributed by Addgene...) are generated from lentiviral preparations (described above) that are subject to a concentration step...

...blood test for HMGB1, the high mobility group box 1 protein. Normally localized to the nucleus, if the...tumors.  This process is not quick. It can take decades from the first exposure to asbestos to the time...T-cells to more easily recognize and cause the destruction of cancer cells. Pembrolizumab has shown some...

...fragment that needs to be amplified should be within 1-10 kB in size. The structure of the primer should ...18-24 bases 40-60% G/C content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer... Protocols Designing Primers How to Design a Primer You may also like...Tips on designing primers for PCR Protocols...Cloning by PCR Agarose Gel Electrophoresis Primer Design for PCR Oligonucleotide primers are necessary when... when running a PCR reaction. One needs to design primers that are complementary to the template region.... They are synthesized chemically by joining nucleotides together. One must selectively block and unblock...

...again. Here’s that process!   Figure 1: The Design-Build-Test-Learn cycle adapted for video making...Good as a WFH desk, and as a video control center.   Cycle 2: Zooming Forward Design: We produced ... So, much like SynBio, it was time to start our Design-Build-Test-Learn cycles. If you’re not familiar...through. I know cameras, but we needed someone else. Design: Jennifer thankfully volunteered, and we decided...here, the operator is in a different room.   Design: The previous cycle made me realize we needed to...restrictions in place, most importantly, masks.  Besides the muffled voices beneath masks, humans rely on...reach for Addgene.  So, onto research! I needed to design a system that would allow me to set up a camera...

...commandments for making it all work – most of the time. #1 Thou shalt find enjoyment in your work If you are ...that extra two miles which is way more fun. And despite those who may wish to tell you so – towel corners...

...first foray into Reddit. Here are some reasons why: 1. Reddit has a large and active community of people...They also host AMA forums where users can ask designated scientists anything about a particular topic....calendar invite over to the r/Science team with a description of our AMA and Joanne’s preferred time to start...was writing all day and straight on through her designated two hours for answering questions. She was able...toxic advisors, fruitless job searches, and even despair at having chosen an academic past. I hope I was...receive via email. The cool features are too many to describe here, but I have over 2,000 notes which Evernote...

...email Ryan at [email protected]   References 1. Larivière, Vincent, Yves Gingras, and Éric Archambault.... After months, or years, of grueling effort to design, fund, and conduct a research project, the vast...provide more than enough time for researchers to describe their science, and for viewers to decide if reading...is of interest. In his research, Scott Spicer concludes that “… the emergence of the video abstract genre...websites). WeShareScience is a social platform that provides links to the videos, keeping the copyright and...

...levels of DnaA to get the job done.   Figure 1: DNA melting by DnaA and IHF is the first step of ...replicate—but not always to the same extent. As described in our previous Origin of Replication post, DNA...replication. For example, the plasmid ColIb-P9 encodes the protein RepZ to drive its own replication. ...

...conference for any reason was a Singleton. Figure 1: The Binning Singletons logo. Image: James et al.,...was that kid who used recycled notebook paper, despite the fact that it was gray. He has worked in molecular...molecular and microbiology for the last two decades as a staff scientist, but before going to graduate ...

... panel design. Find streptavidin plasmids and biotin plasmids at Addgene!     Figure 1: Indirect... this system is increased flexibility for panel design. Another perk is that one primary antibody can ...for example, a previously induced gene knockout. Besides amine- and DNA-binding dyes, nucleoside analogs...walk through the topics we covered in this post. Besides direct labeling, indirect labeling can be used ...surface, dump gating is a useful way to get rid of undesired cell populations. Dump gating uses the same color...

...collaborations, keep these jobs in mind! References 1. Sinche, Melanie, et al. "An evidence-based evaluation...collaborators than you realize. Being at a university provides the perfect platform for practicing initiative...

...complex. So how exactly does this work? Figure 1: APOBEC- and Cas9-mediated editing (ACE) reporter ... plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and subsequent...

...’re not one of these people yourself.  Fig 1: Quote from Michael Ende, courtesy of morefamousquotes.com...even better. The Thomas Kilmann Conflict Model describes 5 basic approaches to conflict resolution based...

...experience in cancer research After graduating with a 2:1 BSc in Molecular Biology (roughly a B average in the...every item I tested was constantly tracked with barcodes and spreadsheets. It was great to be in the lab...industry positions. There were, however, some downsides to working in a large commercial company. In my...aspects of the company and gave me the freedom to design new projects and grow in the role. I understand... job different every day. I love the freedom to design and develop my own projects, but working remotely...

...Bachelor's degree Generally a short-term position (1-2 years) Often a gap-year position taken to transition... are more involved in the science, experimental design, and execution. Can lead to an independent project...advice Give yourself permission to pursue your own desired path. Careers in science are also not limited to...

...of Contents A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids...Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing... References H.1 Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector...information Back to Top A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium The pLKO.1 cloning vector is the backbone...marker encoded in pLKO.1 allows for convenient stable selection. Figure 1 : Map of pLKO.1 containing an shRNA...puromycin should be from 1-10 μg/mL in 1 μg/mL increments. d. Label plates from 1-10 and add appropriate...Appendix I.1. Sequence of pLKO.1 TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning...

...Volume of 1 mg/mL PEI (μL) 1:1 18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9...were transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high.... Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000 Da ...stable. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of OptiPro SFM (per 10 cm plate). 56.7 µL of 1 mg/mL PEI...subclone of HEK293T optimized for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection...to be empirically determined for each new batch of 1 mg/mL PEI prepared. There may be variation between...plasmid using a variety of ratios. Check the cells 1–2 days after transfection to determine what ratio ...

...NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH to 7.0 Autoclave or filter sterilize 1 M sodium phosphate...NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH to 7.0 Autoclave or filter sterilize 1 M of sodium phosphate...protease inhibitor cocktail. Add 1 part Protein A/G binding buffer to 1 part tissue culture supernatant...Choose Option 1 or Option 2 based on the concentration of the pooled sample above. Option 1: Buffer exchange...antibody to 1 mg/mL with PBS if needed. For long term storage, add sterile sodium azide to 1 mM. Option... proceed to Option 1 with a buffer exchange using a Zeba Spin 7 kDa MWCO desalting column. If the concentration...purify recombinant antibodies. Workflow Timeline Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment...

...DNA μL of 1 mg/mL PEI 1:1 18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9 113.4...μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided...: Monday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a...Workflow Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection...inactivated in the lab by heating to 56°C for 30 min. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI) ...to be empirically determined for each new batch of 1 mg/mL PEI and for each cell line. Considerations Before...enough PEI such that the ratio of μg DNA:μg PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid...